金钗石斛生物碱对阻塞型睡眠呼吸暂停低通气综合征的影响

Effects of Dendrobium nobile alkaloids in rats with obstructive sleep apnea syndrome

目的研究金钗石斛生物碱对阻塞型睡眠呼吸暂停低通气综合征(obstructive sleep apnea hyponea syndrome,OSAS)大鼠认知功能、记忆力的影响及其机制。方法从60只SPF级SD雄性大鼠中挑选50只建立OSAS模型,余下10只列入对照组。将成功建模的大鼠随机分为5组:模型组,金钗石斛生物碱高、中、低剂量组和丁苯酞组。金钗石斛生物碱高、中、低剂量组分别用0.36、0.18和0.09 g·kg^(-1)的金钗石斛生物碱灌胃,丁苯酞组用40 mg·kg^(-1)的丁苯酞灌胃,模型组和对照组均用等体积的生理盐水灌胃。每天1次,连续处理12周。通过旷场实验分析大鼠的认知能力;通过新位置识别实验研究大鼠的记忆力;通过苏木素伊红(hematoxylin and eosin,HE)染色观察大鼠海马组织病理学变化;通过原位末端标记法(TdT-mediated dUTP nick and labeling,TUNEL)检测大鼠海马组织细胞的凋亡指数;通过实时荧光逆转录聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测大鼠海马组织中凋亡蛋白酶活化因子1(apoptotic protease-activating factor-1,Apaf-1)、天冬氨酸蛋白水解酶9(cysteinyl aspartate specific proteinase 9,Caspase-9)、Bcl-2和Bax mRNA的相对表达量;通过蛋白免疫印迹法(Western blot,WB)检测大鼠海马组织中Apaf-1、pro-Caspase-9、Cleaved Caspase-9、Bcl-2和Bax蛋白的表达水平。结果大鼠探索总里程模型组长于对照组(P<0.05),丁苯酞组和金钗石斛生物碱高、中、低剂量组均短于模型组(P<0.05);大鼠中央方格停滞时间和大鼠记忆识别指数均表现为模型组低于对照组(P<0.05),丁苯酞组和金钗石斛生物碱高、中、低剂量组均高于模型组(P<0.05);模型组神经细胞数量较少,排列紊乱,神经元出现明显肿胀,核仁消失并出现坏死细胞。丁苯酞组和金钗石斛生物碱高、中、低剂量组均出现好转,其中金钗石斛生物碱高剂量组与对照组较为相似;细胞凋亡指数模型组高于对照组(P<0.05),丁苯酞组和金钗石斛生物碱高、中、低剂量组均低于模型组(P<0.05);Apaf-1、Caspase-9和Bax mRNA,Apaf-1、Cleaved Caspase-9和Bax蛋白相对表达量均表现为模型组均高于对照组(P<0.05),丁苯酞组和金钗石斛生物碱高、中、低剂量组均低于模型组(P<0.05);Bcl-2 mRNA、pro-Caspase-9和Bcl-2蛋白相对表达量模型组低于对照组(P<0.05),丁苯酞组,金钗石斛生物碱高、中、低剂量组均高于模型组(P<0.05)。结论金钗石斛生物碱可改善OSAS大鼠的认知能力和记忆能力,减轻大鼠海马角1(cornus ammonis 1,CA1)区组织损伤,抑制细胞凋亡,推测其机制可能与抑制Apaf-1/Caspase-9信号通路、抑制Caspase-9活化、下调Bax表达水平、上调Bcl-2表达水平有关。

Objective To investigate the effect and mechanism of Dendrobium nobile alkaloids on cognitive function and memory in rats with obstructive sleep apnea syndrome(OSAS).Methods 50 rats were selected to establish the OSAS models from 60 male SPF SD rats.The remaining 10 were recorded as the control group.The successfully modeled rats were randomly divided into 5 groups:model group,Dendrobium nobile alkaloid high,medium,low dose groups and butylphthalide group.Dendrobium nobile alkaloid high,medium and low dose groups were given 0.36,0.18 and 0.09 g·kg^(-1) Dendrobium nobile alkaloids,respectively.The butylphthalide group was given 40 mg·kg^(-1) butylphthalide,once a day,continuous treatment for 12 weeks.The model group and the control group were gavaged with equal volume of normal saline.Rat cognitive abilities were analyzed by open field experiment.Rat memory was analyzed by new position recognition experiment.The histopathological changes of rat hippocampus were observed by using hematoxylin and eosin(HE)staining.Apoptosis indexes of rat hippocampus tissues were detected by TdT-mediated dUTP nick and labeling(TUNEL).The relative mRNA expression levels of apoptotic protease-activating factor-1(Apaf-1),Cysteinyl aspartate specific proteinase 9(Caspase-9),Bcl-2 and Bax in rat hippocampal tissues were measured by real-time quantitative polymerase chain reaction(RT-qPCR).Protein expression levels of Apaf-1,pro-caspase-9,cleaved caspase-9,Bcl-2 and Bax in rat hippocampal tissues were detected by protein Western blot(WB).Results The total mileage of rats explored in the model group was longer than that in the control group(P<0.05),of which the butylphthalide group and Dendrobium nobile alkaloid high,medium and low dose groups were shorter than the model group(P<0.05).The central square stagnation time and the memory recognition index of the model group were lower than those of the control group(P<0.05),and the butylphthalide group and Dendrobium nobile alkaloid high,medium and low dose groups were higher than those of the model group(P<0.05).In the model group,the number of nerve cells was small,the arrangement was disordered,the neurons appeared obvious swelling,the nucleoli disappeared and the necrotic cells appeared.Both the butylphthalide group and the Dendrobium nobile alkaloid high,medium and low dose groups showed improvement,and the Dendrobium nobile alkaloid high dose group was similar to the control group.The apoptosis index of model group was higher than of the control group(P<0.05),of which the butylphthalide group and Dendrobium nobile alkaloid high,medium and low dose groups were lower than that of the model group(P<0.05).The relative expressions of Apaf-1,Caspase-9,Bax mRNA and Apaf-1,cleaved caspase-9 and Bax protein of the model group were higher than those of the control group(P<0.05),of which the butylphthalide group and Dendrobium nobile alkaloid high,medium and low dose groups were lower than those of the model group(P<0.05).The relative expression levels of Bcl-2 mRNA,pro-caspase-9 and Bcl-2 proteins in the model group were lower than those in the control group(P<0.05),of which the butylphthalide group and Dendrobium nobile alkaloids high,medium and low dose groups were higher than those of the model group(P<0.05).Conclusion Dendrobium nobile alkaloids can improve the cognitive and memory abilities of OSAS rats,reduce the tissue damage in the hippocampal cornus ammonis 1(CA1)area of rats,and inhibit apoptosis.It is speculated that the mechanism may be related to inhibiting the Apaf-1/Caspase-9 signaling pathway,inhibiting the activation of Caspase-9,down-regulating the expression level of Bax,and up-regulating the expression level of Bcl-2.